Patent Application
20160235837
The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 16, 2014, is named 13795-48876_SL.txt and is 174,231 bytes in size.
The present invention relates to therapies for preventing and treating Middle East Respiratory Syndrome Virus (MERS CoV), methods of differentiating lethality of MERS CoV and of predicting outbreaks of lethal MERS CoV, and compounds for diagnostic, therapeutic, and/or preventive purposes in MERS CoV.
Middle East Respiratory Syndrome (MERS) is a viral respiratory illness first reported in Saudi Arabia in 2012. The disease is caused by a coronavirus known as MERS-CoV. Most confirmed MERS-CoV infections manifest as severe acute respiratory illness with fever, cough, and shortness of breath. Mortality rate in confirmed cases is around fifty percent. At the present time, confirmed cases are linked to four countries in or near the Arabian Peninsula including, Saudi Arabia, Jordan, and Qatar. Spread of the virus from ill people to others through close contact has reportedly been observed in a few cases. Nevertheless, at present the virus has not been observed to spread in a sustained way in human communities.
MERS CoV is a novel coronavirus belonging to the genus Betacoronavirus. Severe Acute Respiratory Coronavirus (SARS-CoV) also belongs to this genus. MERS CoV has been referred to as a SARS-like virus but is nevertheless more closely related to the bat coronaviruses HKU4 and HKU5 (lineage 2C) than it is to SARS-CoV (lineage 2B) and shares more than 90% sequence identity in closest relationships with HKU4 and HKU5.
MERS CoV was first reported Sep. 24, 2012 from Saudi Arabia. The virus was isolated and identified from the lungs of a 60-year-old male patient with acute pneumonia and acute renal failure.
MERS CoV is a single-stranded RNA Group IV virus. It is positive sense. The virus is categorized in Order Nidovirales, Family Coronaviridae, Subfamily Coronavirinae, and Genus Betacoronavirus. Species within the genus include Betacoronavirus 1 (commonly called Human coronavirus OC43), Human coronavirus HKU1, Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus, Tylonycteris bat coronavirus HKU4, and MERS-CoV. The MERS CoV genome has been reported to contain the following gene segments: N, M, S, ORF4b, ORF1ab, ORF1a, ORF1b, NS3C, and N3.
In April 2012, six hospital workers were diagnosed with acute respiratory failure of unknown origin. Of the six, two died. All the cases were reported to the European Centre for Disease Prevention and Control (ECDC). Following publication of the MERS CoV strain, a trace back was undertaken. Epidemiologists discovered the Jordan cases. Using stored laboratory samples for all six, it was found that samples from the two patients who had died tested positive for MERS CoV.
On May 14, 2014, Bloomberg news reported that at least 365 cases of MERS had been reported to the World Health Organization since Mar. 27, 2014, which was reported as more than double the number of infections since the start of the outbreak. It reported that contact with camels during the spring, when females wean their young, was thought to have contributed to the surge in new cases, and this was thought to have been amplified by poor infection control measures in hospitals. An analysis of 128 cases in the Saudi city of Jeddah had found that more than 60 percent were infected while in hospital. Bloomberg News, Simeon Bennett, 9:25 a.m.
There is a need in the art for quantitative methods of predicting further progression and additional outbreaks or waning of MERS CoV. There is likewise a need in the art for methods of preventing, and treating MERS CoV infections and outbreaks and a need for therapies against MERS CoV including prophylactic therapies, such as vaccines, and treatments, such as therapies for providing passive immunity and other methods of blocking progression or transmission of MERS CoV and related viruses.
Replikin peptides are a family of small peptides that have been correlated with the phenomenon of rapid replication in SARS, influenza, malaria, West Nile virus, foot and mouth disease, and many other pathogens. See, e.g., WO 2008/143717. Replikin peptides have likewise been generally correlated with the phenomenon of rapid replication in viruses, organisms, and malignancies.
Identification of Replikin peptides has provided targets for detection and treatment of pathogens, including vaccine development against virulent pathogens such as influenza virus, malaria, West Nile virus, and foot and mouth disease virus. See, e.g., WO 2008/143717. In general, knowledge of and identification of this family of peptides enables development of effective therapies and vaccines for any pathogen that harbors Replikins. The phenomenon of the association of Replikins with rapid replication and virulence has been fully described in U.S. Pat. No. 7,189,800; U.S. Pat. No. 7,176,275; U.S. Pat. No. 7,442,761; U.S. Pat. No. 7,894,999, U.S. Pat. No. 8,050,871, and U.S. application Ser. No. 12/108,458. Both Replikin concentration (number of Replikins per 100 amino acids) and Replikin composition have been correlated with the functional phenomenon of rapid replication.
There is a continuing need for monitoring Replikin sequences in MERS CoV to identify compounds for therapies that target MERS CoV. There is also a need to develop Replikin-based therapies that are effective across strains and within strains as they mutate over time.
In response to these continuing needs and despite extensive efforts to understand infectivity and lethality in MERS CoV and to track and predict outbreaks of MERS CoV, applicants have now surprisingly applied their previous discovery of Replikin chemistry in the virus genome structure to methods of predicting outbreaks of MERS CoV. They have likewise now surprisingly provided methods of identifying conserved targets in emerging MERS CoV against which vaccines are provided and likewise may be provided prior to or at the outset of any further outbreak. Such vaccine development can be undertaken in as few as seven days.
The present invention provides compounds for diagnostic, therapeutic, and/or preventive purposes against MERS CoV and methods of predicting outbreaks of MERS CoV.
A first non-limiting aspect of the present invention provides an isolated or synthesized protein fragment or peptide comprising at least one peptide sequence that is at least 50% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, the at least one peptide sequence may be at least 80% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, the at least one peptide sequence may be at least 46% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, the at least one peptide sequence may be at least 53% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, the at least one peptide sequence may be at least 60%, 70%, 80%, 90%, or 95% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In another non-limiting embodiment, the isolated or synthesized protein fragment or peptide may comprise at least one Replikin peptide sequence identified in an MERS CoV or at least one homologue of the at least one Replikin sequence identified in an MERS CoV.
In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of at least one peptide sequence that is at least 50% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of at least one peptide sequence that is at least 46%, 50%, 53%, 60%, 70%, 80%, 90%, or 95% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, the at least one Replikin peptide sequence identified in a MERS CoV is at least one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide consists of up to 50, up to 75, up to 100, up to 150, or up to 200 or more amino acid residues.
In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of at least one peptide sequence that is at least 80% homologous with at least one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide consists essentially of at least one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide consists of at least one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide consists of at least one of SEQ ID NO(s): 1-28. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide of consists of at least one of SEQ ID NO(s): 1-9. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide of consists of at least one of SEQ ID NO(s): 5, 18, 22, and 23. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide consists of at least one of SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28.
In a non-limiting embodiment an isolated or synthesized protein fragment or peptide consists of up to 200 amino acid residues. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of up to 150 amino acid residues. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of up to 100 amino acid residues. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of up to 75 amino acid residues. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of up to 50 amino acid residues. In a non-limiting embodiment, an isolated or synthesized protein fragment or peptide consists of up to 25 amino acid residues.
An isolated or synthesized protein fragment or peptide may consist essentially of at least one Replikin peptide sequence identified in an MERS CoV or at least one homologue of said at least one Replikin peptide sequence identified in an MERS CoV. The isolated or synthesized protein fragment or peptide may consist of at least one Replikin peptide sequence identified in an MERS CoV or at least one homologue of said at least one Replikin peptide sequence identified in an MERS CoV. Another non-limiting embodiment provides an isolated or synthesized peptide sequence comprising at least one functional fragment of a Replikin sequence identified in MERS CoV. In a non-limiting embodiment, the protein fragment or peptide may be isolated or derived from one of the gene segments of the genome of MERS CoV. A non-limiting gene segment may be N, M, S, ORF4b, ORF1ab, ORF1a, ORF1b, NS3C, N3, or any other gene segment.
In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide may comprise at least one Replikin peptide sequence of KQKAPKH (SEQ ID NO: 1), KSAGHPFNK (SEQ ID NO: 2), KRSHSPTKK (SEQ ID NO: 3), HSPTKKLRYVK (SEQ ID NO: 4), KARKRSHSPTK (SEQ ID NO: 5), KHVEVFTDGK (SEQ ID NO: 6), HKWKMVVCDK (SEQ ID NO: 7), KKHVEVFTDGK (SEQ ID NO: 8), KDAAAAKNKMRHK (SEQ ID NO: 9), KSVVRHLGVTK (SEQ ID NO: 10), KFYQHVINGCK (SEQ ID NO: 11), KQVHQVQLTDK (SEQ ID NO: 12), KGDSCSSNCKH (SEQ ID NO: 13), HARLKGGLILK (SEQ ID NO: 14), KAMLLKKEPLLYVPIRLAGH (SEQ ID NO: 15), KHLVPLMHK (SEQ ID NO: 16), KYYAFLNKH (SEQ ID NO: 17), KAAVHKWK (SEQ ID NO: 18), KLNPSEDFIKH (SEQ ID NO: 19), KFCDHMVK (SEQ ID NO: 20), KPGHAMPSLFK (SEQ ID NO: 21), KNKMRHK (SEQ ID NO: 22), KRSHSPTK (SEQ ID NO: 23), KDAAAAKNKMRH (SEQ ID NO: 24), KMRHKRTSTK (SEQ ID NO: 25), HVERKDVPYPK (SEQ ID NO: 26), KGMQLLHTK (SEQ ID NO: 27), or KEGSSVTLKH (SEQ ID NO: 28), or at least one homologue of SEQ ID NO(s): 1-28. In a non-limiting embodiment, the isolated or synthesized protein fragment or peptide may consists essentially of at least one Replikin peptide sequence of SEQ ID NO(s): 1-28 or at least one homologue of SEQ ID NO(s): 1-28. In another non-limiting embodiment, the isolated or synthesized protein fragment or peptide may consist of at least one Replikin peptide sequence of SEQ ID NO(s): 1-28 or at least one homologue of SEQ ID NO(s): 1-28. Another non-limiting embodiment provides an isolated or synthesized peptide sequence comprising at least one functional fragment of at least one Replikin peptide sequence of SEQ ID NO(s): 1-28. Another non-limiting embodiment provides an isolated or synthesized peptide sequence comprising at least one functional fragment of at least one Replikin peptide sequence of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, 237, or 238.
A non-limiting embodiment of the first aspect of the invention provides an isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising at least one Replikin peptide of an MERS CoV virus. A further embodiment of the first aspect of the invention provides an isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising at least one peptide sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 100%, homologous with at least one Replikin peptide sequence identified in a MERS CoV virus. In a non-limiting embodiment, the at least one sequence is one of SEQ ID NO(s): 1-28.
In a further non-limiting embodiment of the first aspect of the present invention, the isolated or synthesized protein, protein fragment, polypeptide, or peptide consists of 7 to about 50 amino acid residues comprising at least one peptide A, wherein said peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, homologous with at least one Replikin peptide sequence identified in an MERS CoV. In one non-limiting embodiment, said at least one Replikin peptide sequence identified in MERS CoV is at least one peptide sequence of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239, or SEQ ID NO(s): 1-28, SEQ ID NO(s): 1-9, SEQ ID NO(s): 5, 18, 22, and 23, or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28.
In a further non-limiting embodiment of the first aspect of the present invention, the isolated or synthesized protein, protein fragment, polypeptide, or peptide consists essentially of a Replikin peptide identified in MERS CoV. In a further non-limiting embodiment, the Replikin peptide sequence identified in an MERS CoV is at least one peptide sequence of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239.
In a non-limiting embodiment, the Replikin sequence is shared among isolates of MERS CoV isolated from camel and human. In a further non-limiting embodiment, the Replikin sequences shared among human and camel isolates are identical. In a further non-limiting embodiment, the lysine and histidine residues that make the Replikin sequences are identical among human and camel isolates while one or more other amino acid residues are not identical. In non-limiting embodiment, the Replikin sequences are conserved in camels and humans over one, two, three, or more years.
Another non-limiting embodiment of the first aspect of the invention provides a biosynthetic composition comprising the protein, protein fragment, polypeptide, or peptide of an aspect of the invention. In a further non-limiting embodiment, the biosynthetic composition consists essentially of a Replikin peptide of MERS CoV or consists of a Replikin peptide of MERS CoV. In a non-limiting embodiment, an isolated protein, protein fragment, polypeptide, or peptide is chemically synthesized by solid phase methods.
A second non-limiting aspect of the present invention provides an immunogenic and/or blocking composition comprising at least one protein, protein fragment, polypeptide, or peptide of any one of the above-listed proteins, protein fragments, polypeptides, or peptides including and not limited to comprising at least one Replikin peptide sequence identified in MERS CoV or at least one homologue of said at least one Replikin peptide identified in MERS CoV or at least one functional fragment of at least one Replikin peptide sequence identified in MERS CoV. In a non-limiting embodiment of the second aspect of the present invention, the immunogenic and/or blocking composition comprises at least one peptide sequence of SEQ ID NO(s): 1-28. In a further non-limiting embodiment, the immunogenic and/or blocking composition comprises at least one peptide consisting essentially of any one of SEQ ID NO(s): 1-28. In further non-limiting embodiment, the immunogenic and/or blocking composition comprises at least one peptide consisting of any one of SEQ ID NO(s): 1-28 or at least one functional fragment of any one of SEQ ID NO(s): 1-28. In a non-limiting embodiment, the Replikin sequence is shared among isolates isolated from camel and human. In a further non-limiting embodiment, the Replikin sequences are identical. In non-limiting embodiment, the Replikin sequences are conserved in camels and humans over one, two, three, or more years.
A third non-limiting aspect of the present invention provides a vaccine comprising at least one protein, protein fragment, polypeptide, or peptide comprising or consisting of a Replikin sequence or homologue thereof including any one of the above-listed proteins, protein fragments, polypeptides, or peptides. In a non-limiting embodiment, a Replikin sequence is shared among isolates isolated from camel and human. In a further non-limiting embodiment, the Replikin sequences are identical. In a further non-limiting embodiment, the lysine and histidine residues that make the Replikin sequences are identical among human and camel isolates while one or more other amino acid residues are not identical. In a non-limiting embodiment, the Replikin sequences are conserved in camels and humans over one, two, three, or more years.
In a non-limiting embodiment of the third aspect of the present invention, the vaccine comprises at least one peptide sequence of any one of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28, comprises at least one peptide sequence consisting essentially of any one of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28, and/or comprises at least one peptide sequence consisting of any one of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28, or at least one functional fragment of any one of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28, at least one functional fragment of a Replikin peptide sequence identified in MERS CoV, or at least one functional fragment of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28.
In a further non-limiting embodiment of the third aspect of the present invention, the vaccine comprises a mixture of a plurality peptides of different sequences, wherein each of said peptides of different sequences is at least 80% homologous with at least one Replikin sequence identified in MERS CoV. In a non-limiting embodiment, the vaccine comprises a mixture of a plurality of peptides of different sequences, wherein at least one peptide of the mixture of a plurality of peptides of different sequences is at least 80% homologous with each of SEQ ID NO(s): 1-9, each of SEQ ID NO(s): 1-28, each of SEQ ID NO(s): 5, 18, 22, and 23, or each of SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a non-limiting embodiment, the vaccine comprises a mixture of a plurality of peptides of different sequences where at least one peptide of the mixture of a plurality of peptides of different sequences consists of each of SEQ ID NO(s): 1-9, each of SEQ ID NO(s): 1-28, each of SEQ ID NO(s): 5, 18, 22, and 23, or each of SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a non-limiting embodiment, a vaccine comprises at least one functional fragment of any one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, a vaccine comprises a pharmaceutically-acceptable carrier and/or adjuvant.
In a non-limiting embodiment, a vaccine comprises a mixture of a plurality of peptide sequences of any of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28 and/or a mixture of a plurality of homologues of peptide sequences of any of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the vaccine comprises a mixture of a plurality of peptide sequences consisting essentially of any one or more of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the vaccine comprises a mixture of a plurality of peptide sequences consisting of any one or more of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the vaccine comprises a mixture of a plurality of peptides consisting of each of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28, or any combination of the listed sequences.
In another non-limiting embodiment of the third aspect of the invention, the vaccine comprises a mixture of Replikin peptides. In a non-limiting embodiment, the vaccine comprises an approximately equal molar mixture of the isolated or synthesized peptides of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the vaccine comprises approximately equal weight of the isolated or synthesized peptides of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the vaccine comprises a pharmaceutically-acceptable carrier and/or adjuvant. In a further non-limiting embodiment, the vaccine comprises peptides in sterile water. In a further non-limiting embodiment, the vaccine comprises freeze-dried peptides in sterile water. In a further non-limiting embodiment, the vaccine is for the treatment or prevention of MERS CoV infection.
A fourth non-limiting aspect of the invention provides an isolated or purified antibody, antibody fragment, or binding agent that specifically binds to at least a portion of a protein fragment or peptide comprising at least one peptide sequence that is at least 80% homologous with at least one Replikin peptide sequence identified in a MERS CoV. In a non-limiting embodiment, the isolate or purified antibody, antibody fragment, or binding agent specifically binds at least a portion of any one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, the isolate or purified antibody, antibody fragment, or binding agent specifically binds at least a portion of any one of SEQ ID NO(s): 1-28. In a non-limiting embodiment, the isolate or purified antibody, antibody fragment, or binding agent specifically binds at least a portion of any one of SEQ ID NO(s): 1-9. In a non-limiting embodiment, the isolate or purified antibody, antibody fragment, or binding agent specifically binds at least a portion of any one of SEQ ID NO(s): 5, 18, 22, and 23 or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28.
A fourth non-limiting aspect of the invention provides an isolated or purified antibody, antibody fragment, or binding agent that binds to at least a portion of an amino acid sequence of at least one protein, protein fragment, polypeptide, or peptide comprising a peptide sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one Replikin peptide sequence identified in MERS COV. In a further embodiment, the at least one Replikin peptide sequence identified in MERS COV is at least one peptide sequence of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28.
A fifth non-limiting aspect of the present invention provides a method of making a vaccine comprising: isolating or synthesizing a protein fragment or peptide comprising at least one peptide sequence that is at least 80% homologous with at least one Replikin peptide sequence identified in a MERS CoV as a component of a vaccine; and making the vaccine with the component.
A non-limiting embodiment comprises: selecting at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising at least one peptide sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100% homologous with at least one Replikin peptide sequence identified in MERS CoV as a component of a vaccine; and making said vaccine. In a non-limiting embodiment, the method of making a vaccine comprises selecting at least one isolated or synthesized peptide of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28 as at least one component and making said vaccine with the at least one component.
In another non-limiting embodiment, the method of making a vaccine comprises selecting at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or up to 28 or more isolated or synthesized Replikin peptide sequences identified in MERS CoV and/or isolated or synthesized functional fragments of Replikin peptide sequences identified in MERS CoV and/or homologous of Replikin peptide sequences identified in MERS CoV. In a further embodiment, the isolated or synthesized Replikin peptide sequences, functional fragments of Replikin peptide sequences identified in MERS CoV, or homologues of Replikin peptide sequences identified in MERS CoV comprise at least one peptide sequence of SEQ ID NO(s): 1-28, at least one functional fragment of at least one peptide sequence of SEQ ID NO(s): 1-28, at least one functional fragment of at least one Replikin peptide sequence identified in MERS CoV, or at least one homologues of at least one Replikin peptide sequence identified in MERS CoV. In another non-limiting embodiment, the at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide has the same amino acid sequence as at least one protein, protein fragment, polypeptide or peptide identified in a relatively lethal strain of MERS CoV up to seven days, one month, six months, one year, two years, or three years prior to making said vaccine.
A sixth non-limiting aspect of the present invention provides a method for preventing or treating MERS COV infection comprising administering at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising at least one peptide sequence to an animal or human, where the peptide sequence is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one Replikin peptide identified in MERS CoV. In a further non-limiting embodiment, the Replikin peptide sequence is at least one peptide sequence of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the Replikin peptide sequence is at least one peptide sequence of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. In a non-limiting embodiment, the at least one isolated or synthesized protein fragment, polypeptide, or peptide consists of at least one peptide sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptide sequences of SEQ ID NO(s): 1-28. In another non-limiting embodiment, the at least one isolated or synthesized peptide of SEQ ID NO(s): 1-28 is administered to a human or other animal. In a further non-limiting embodiment the at least one Replikin peptide sequence is at least one peptide sequence of SEQ ID NO(s): 1-28.
A non-limiting embodiment of the sixth aspect provides use of at least one Replikin peptide sequence identified in MERS CoV, at least one homologue of at least one Replikin peptide sequence identified in MERS CoV, or at least one functional fragment of at least one Replikin peptide sequence identified in MERS CoV in the manufacture of a medicament for preventing or treating MERS CoV infection. In a non-limiting embodiment, the Replikin peptide sequence is any one of SEQ ID NO(s): 1-28, 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. A non-limiting embodiment provides use of a Replikin sequence, a homologue of a Replikin sequence, or a functional fragment of a Replikin sequence of MERS CoV for the prevention or treatment of MERS CoV.
A seventh non-limiting aspect of the present invention provides a method of predicting expansion or retraction of a population of MERS CoV comprising, respectively, identifying an increase in the percentage of isolates of MERS CoV having a Replikin concentration (number of Replikin sequences per 100 amino acid residues) in at least one portion of the genome greater than 4.0 between two time periods or identifying a decrease in the percentage of isolates of MERS CoV having a Replikin concentration (number of Replikin sequences per 100 amino acid residues) in at least one portion of the genome greater than 4.0 between two time periods. In a non-limiting embodiment, the more than two time periods are compared and the percentage of isolates in the more than two time periods shows a pattern of increase or decrease. In a non-limiting embodiment, the portion of the genome reflects encoding of an expressed protein. In a non-limiting embodiment, the portion of the genome is N, M, S, ORF4b, ORF1ab, ORF1a, ORF1b, NS3C, N3, or any other gene segment of the genome.
A non-limiting embodiment of the seventh aspect of the present invention provides a method of differentiating between relatively more lethal and relatively less lethal forms of MERS CoV. A non-limiting embodiment provides a method of identifying and/or diagnosing a relatively more lethal form of MERS CoV comprising determining the Replikin concentration of at least one portion of at least one protein of at least one isolate of MERS CoV or at least one portion of at least one gene that expresses at least one protein of the at least one isolate of MERS CoV and comparing the Replikin concentration of the at least one isolate of MERS CoV to a comparable Replikin concentration in at least one other isolate of MERS CoV. In a further non-limiting embodiment, the at least one portion of at least one protein comprises the entirety of at least one protein expressed in MERS CoV and the comparable Replikin concentration is the Replikin concentration of the entirety of the same protein expressed in MERS CoV from the at least one other isolate of MERS CoV. In a non-limiting embodiment, the Replikin concentration of the at least one isolate of MERS CoV is a mean of Replikin concentrations determined in a plurality of isolates of MERS CoV. In a further non-limiting embodiment, the Replikin concentration of the at least one other isolate of MERS CoV is a mean of Replikin concentrations determined in a plurality of other isolates of MERS CoV. In a further non-limiting embodiment, the plurality of isolates of MERS CoV is a collection of isolates isolated in a given year and the plurality of other isolates of MERS COV is a collection of isolates isolated in a different year. In a further non-limiting embodiment, the Replikin concentration of the more lethal isolate of MERS COV is 3.0 or greater, 4.0 or greater, or 5.0 or greater per 100 amino acid residues. In a further non-limiting embodiment, the Replikin concentration of the more lethal isolate of MERS COV is 4.0 or greater per 100 amino acid residues. In a further non-limiting embodiment, the Replikin concentration of the more lethal isolate of MERS COV is 4.6 per 100 amino acid residues or greater. In a further non-limiting embodiment, a vaccine is manufactured following the differentiation between relatively more lethal and relatively less lethal forms of MERS CoV. In a further non-limiting embodiment, the vaccine comprises at least one structure of the isolate of MERS CoV differentiated as relatively more lethal. In a further non-limiting embodiment, the vaccine comprises at least one Replikin peptide sequence identified in the isolate of MERS CoV differentiated as relatively more lethal.
In a further non-limiting embodiment of the seventh aspect of the present invention, the at least one portion of at least one gene expressing at least one protein is at least one portion of the ORF4b gene region.
In a further non-limiting embodiment of the seventh aspect of the present invention, the Replikin concentration of the at least one isolate of MERS CoV is greater than the Replikin concentration of the at least one other isolate of MERS CoV. In a further non-limiting embodiment the Replikin concentration is a mean Replikin concentration of a plurality of isolates with standard deviation from the mean and the standard deviation from the mean is greater than the standard deviation from the mean Replikin concentration of a plurality of other isolates.
Another non-limiting embodiment of the seventh aspect of the invention provides a method of determining an increased probability of an outbreak of MERS CoV within about one year following an increase in Replikin concentration in an isolate of MERS CoV comprising identifying an increase in the concentration of Replikin sequences in at least one first isolate of MERS CoV as compared to at least one other isolate of MERS CoV wherein said at least one first isolate is isolated at a later time period than said one other isolate and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of MERS CoV within about one year following the increase in the concentration of Replikin sequences.
In a non-limiting embodiment, a method of prediction comprises: (1) obtaining a plurality of isolates of MERS CoV wherein at least one of said isolates is isolated about six months to about 3 years later than at least one other of said isolates; (2) analyzing the amino acid sequence of at least one protein or protein fragment in each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (3) comparing the concentrations of Replikin sequences in the at least one protein or protein fragment in each isolate of the plurality of isolates one to another; (4) identifying an increase in the concentration of Replikin sequences in said plurality of isolates over at least one time period of about six months or greater; and (5) predicting an outbreak of MERS CoV within about one month to about three years following said identified increase in the concentration of Replikin sequences. In another embodiment of the invention, the outbreak of MERS CoV is predicted within about six months. In a further embodiment of the invention, the outbreak of MERS CoV is predicted within about one year to about three years. In a further non-limiting embodiment, the method of prediction further comprises processing at least one step of the method on a computer.
In a further non-limiting embodiment of the seventh aspect of the invention, the method of prediction further comprises comparison of the standard deviation from the mean of Replikin concentrations of isolates of MERS CoV from a given time period, such as a given month, a given year, or any other given time period. In a further non-limiting embodiment, the Replikin concentration is a mean Replikin concentration of a plurality of isolates with standard deviation from the mean and the standard deviation from the mean is greater than the standard deviation from the mean Replikin concentration of a plurality of other isolates.
A further non-limiting embodiment 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 MERS CoV or an increase in virulence, morbidity, and/or mortality of MERS CoV. 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 MERS CoV 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.
Another non-limiting embodiment of the seventh aspect of the invention provides a computer system, including a processor coupled to a network and a memory coupled to the processor, the memory containing a plurality of instructions to perform a method of predicting an increase in virulence, morbidity, or mortality as compared to at least one second malignancy.
Another non-limiting embodiment of the seventh aspect of the invention provides a machine-readable storage medium having stored thereon executable instructions that, when executed by a processor, cause the processor to provide sufficient data to a user, a display, or a printout such that said user or a user of said display or said printout may predict an increase in virulence, morbidity, or mortality of MERS CoV based on regression analysis. Another non-limiting embodiment provides a computer system, comprising: a processor coupled to a network; a memory coupled to the processor, the memory containing a plurality of instructions to perform the method of predicting an increase in virulence, morbidity, or mortality of MERS CoV based on the regression analysis.
A non-limiting embodiment provides an electromagnetic signal carrying computer-readable instructions for performing a method of predicting an outbreak of MERS CoV or the relative lethality or virulence of MERS CoV. In a non-limiting embodiment, the signal is non-transient.
A “protein fragment” as used in this specification is any fragment of an expressed whole protein, which is any portion of an expressed whole protein where a “portion” of a protein is less than an expressed whole protein. A protein fragment reflects an expressed whole protein with one or more amino acids removed from the amino acid sequence of the expressed whole protein. A protein fragment may also reflect an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or greater homologous with any portion of an expressed whole protein. A “polypeptide,” as used in this specification, is any portion of a protein fragment and is less than an expressed whole protein.
A “whole protein” or an “expressed whole protein” as used in this specification reflect a protein that is expressible from an intact gene of MERS CoV from a start codon to a stop codon. A whole protein or expressed whole protein may also reflect a whole protein or expressed whole protein that has been subject to cellular processing to create a protein that is capable of functioning within the virus replication system in a proper manner for virus replication. A protein fragment, polypeptide, or peptide “partially matches” the amino acid sequence of an expressed whole protein when the protein fragment, polypeptide, or peptide shares substantial homology with at least a portion of the expressed whole protein.
A “functional fragment” of a Replikin sequence as described herein is a fragment, variant, analog, or chemical derivative of a Replikin sequence that retains at least a portion of the immunological cross reactivity with an antibody specific for the Replikin sequence. A fragment of the Replikin sequence refers to any subset of the molecule. Variant peptides of the sequence may be made by direct chemical synthesis, for example, using methods well known in the art. An analog of a Replikin sequence to a non-natural protein or polypeptide is substantially similar to either the Replikin sequence of the protein or a fragment thereof. Chemical derivatives of a Replikin sequence contain additional chemical moieties.
As used herein, the term “preferentially binds” or “specifically binds” and related terms referencing the interaction of a binding molecule such as, for example, an antibody, and the structure to which it binds (antigen) means that the binding molecule preferentially recognizes the structure to which it binds even when present among other molecules (such as in a mixture of molecules). Specific or preferential binding of a binding molecule to a binding structure or an immunogenic portion of a binding structure is specific and preferential when the binding molecule binds to the structure or portion thereof and does not bind with the same level of affinity to other structures. Binding affinity may be determined by one of ordinary skill in the art using, for example, BIACORE, enzyme-linked immunosorbent assays, or radioimmuno assays. A binding molecule may cross-react with related antigens and preferably does not cross-react with affinity to unrelated antigens. Binding between a binding molecule and the structure to which it binds may be mediated by covalent or non-covalent attachment, or both.
As used herein a “vaccine” is any substance, compound, composition, mixture, or other therapeutic substance that, when administered to a human or animal via any method of administration known to the skilled artisan now or hereafter, produces an immune response, an antibody response, a blocking response, or a protective effect in the human or animal.
As used herein, a “Replikin sequence” is an amino acid sequence of 7 to 50 amino acid residues comprising (1) a first lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues, where the sequence is the shortest sequence comprising the first and second lysine residues of element (1) and the at least one histidine of element (2). A Replikin sequence may comprise more than two lysine residues and more than one histidine residue so long as at least two of the lysine residues and at least one histidine residue reflect the requirements of the definition of a Replikin sequence. A Replikin sequence may be targeted by the immune system within a peptide sequence that comprises more than the shortest sequence comprising the lysine and histidine residues required for a Replikin sequence.
The term “Replikin sequence” can also refer to a nucleic acid sequence encoding an amino acid sequence having 7 to about 50 amino acids comprising:
In polypeptides, peptides, proteins, and protein fragments, the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond forming a compound that is a chain of amino acid residues.
As used herein, a “synthesized” peptide may be synthesized by organic chemical methods and may be synthesized by biosynthetic methods. An “isolated” peptide may refer 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. 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 may be synthesized or isolated as compounds. An isolated peptide may be synthesized by biosynthetic or organic chemical methods.
Proteins, protein fragments, polypeptides, or peptides in this specification may be chemically synthesized by any method known to one of skill in the art now and hereafter. For example, isolated proteins, protein fragments, polypeptides, or peptides may be synthesized by solid phase synthesis. The production of these materials by chemical synthesis avoids the inclusion of (or the need to remove by purification) materials that are byproducts of other production methods such as recombinant expression or isolation from biological material. Such byproducts may include, for example, avian proteins associated with vaccines produced using birds' eggs, bacterial proteins associated with recombinant production in bacteria, or proteins or contaminants associated with any recombinant activity such as with productions of proteins or other sequences in insect cells.
An “encoded” or “expressed” protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of ordinary skill in the art now or hereafter. It should be noted that it is well known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence. As will be understood by one of ordinary 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.
“Homologous” or “homology” or “sequence identity” as used in this specification indicate that an amino acid sequence or nucleic acid sequence exhibits substantial structural equivalence with another sequence, namely, any Replikin peptide sequence (including SEQ ID NO(s): 1-28) identified in an isolate of MERS CoV or any nucleotide sequence encoding a Replikin peptide sequence in an isolate of MERS CoV (a redundancy in a coding sequence may be considered identical to a sequence encoding the same amino acid). To determine the percent identity or percent homology of an identified sequence, a sequence is aligned for optimal comparison purposes with any one of possible basis sequences. For purposes of this paragraph, a basis sequence is a Replikin sequence identified in an isolate of MERS CoV. Where gaps are necessary to provide optimal alignment, gaps may be introduced in the identified sequence or in the basis sequence. When a position in the identified sequence is occupied by the same amino acid residue or same nucleotide as the corresponding position in the basis sequence, the molecules are considered identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). To determine percent homology, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared between the identified (reference) sequence and the basis sequence. The total number of amino acid residues or nucleotides in the identified sequence that are identical with amino acid residues or nucleotides in the basis sequence is divided by the total number of residues or nucleotides in the basis sequence (if the number of residues or nucleotides in the basis sequence is greater than the total number of residues or nucleotides in the identified sequence) with gaps included or by the total number of amino acid residues or nucleotides in the identified sequence (if the number of residues or nucleotides in the identified sequence is greater than the total number of residues or nucleotides in the basis sequence) with gaps included. The final number is determined as a percentage. As such, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps (where a gap must be introduced for optimal alignment of the two sequences) and the length of each gap. Any structural or functional differences between sequences having sequence identity or homology will not affect the ability of the sequence to function as indicated in the desired application.
For example, SEQ ID NO: 6 (KHVEVFTDGK) is considered more than 90% homologous with SEQ ID NO: 8 (KKHVEVFTDGK). The more than 90% homology between SEQ ID NO: 6 and SEQ ID NO: 8 is determined as follows: SEQ ID NO: 6 is the identified sequence. SEQ ID NO: 8 is the basis sequence. Upon alignment, SEQ ID NO: 6 is identical to SEQ ID NO: 8 in all 11 residues of SEQ ID NO: 8 with the exception of the first lysine in SEQ ID NO: 8. To determine percent homology, then, the 10 aligned identical residues are divided by the total number of residues in SEQ ID NO: 8, namely 11 residues, giving 0.909 or greater than 90% homology.
In a further example, SEQ ID NO: 24 (KDAAAAKNKMRH) is more than 46% homologous with SEQ ID NO: 22 (KNKMRHK). SEQ ID NO: 24 is the basis sequence and has 12 residues. SEQ ID NO: 22 is the identified (or reference) sequence and has 7 residues. Six of the seven residues in SEQ ID NO: 22 are in the same position as six residues in SEQ ID NO: 24 when SEQ ID NO: 22 and SEQ ID NO: 24 are optimally aligned. A gap of one residue on the C-terminal end of SEQ ID NO: 24 must be added to encompass the lysine at the C-terminus of SEQ ID NO: 22. Including the gap of one residue on the C-terminal end, SEQ ID NO: 24 has a length of 13 residues. To determine percent homology, then, the 6 aligned identical residues are divided by the total number of residues in SEQ ID NO: 24 (with gap include), namely, 13 residues, giving 0.461 or more than 46% homology.
In another examples, SEQ ID NO: 22 (KNKMRHK) is more than 53% homologous with SEQ ID NO: 9 (KDAAAAKNKMRHK) because the seven residues of SEQ ID NO: 22 align exactly with seven residues of SEQ ID NO: 9 and SEQ ID NO: 9 has 13 total residues (giving greater than 53% homology).
Concerning gaps, the number of gaps in either the basis sequence or the identified sequence should be limited to the number of gaps allowable without significantly compromising the function of the identified sequence as compared to the basis sequence. In general, many gaps in the sequence of the basis peptide or in the sequence of the identified peptide are allowed based on homology as defined herein. Relatively more gaps are allowed if the lysines and histidines that create the definition of the Replikin peptide are identically shared between the basis peptide and the identified peptide. Relatively more gaps are also allowed if the lysines and histidines that create the definition of the Replikin peptide are shared at least in close position (for example within ten, nine, eight, seven, six, five, four, three, two, or one amino acid residue). If some of the lysine residues and histidine residues that create the definition of the Replikin peptide are not present in the identified peptide, fewer gaps may be allowed. Nevertheless, if the identified peptide functions similarly to the basis peptide, any number of gaps is allowed. In general, three or more gaps are allowed in the sequence of the basis peptide or in the sequence of the identified peptide within ten amino acid residues of the basis peptide if no lysines or histidines are present in the identified peptide. Two or more gaps or one or more gaps are also allowed. Nevertheless, if the identified sequence provides the same or a similar function to the basis sequence, more gaps are allowed up to the number of gaps that will provide a homology of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more homology. Additionally, where the lysines and histidines of the Replikin definition are present in both the identified peptide and the basis peptide, there should be no limit on how many gaps are allowed.
The presence of lysines and histidines providing for the Replikin definition in an identified peptide requires significantly less homology because the lysines and the histidines of the Replikin definition provide for conservation of Replikin function. For example, in Table 8 and the description thereof in columns 62 and 63 in U.S. Pat. No. 7,442,761, a highly-mutable tat protein in HIV is described and analyzed. As may be seen from Table 8 in U.S. Pat. No. 7,442,761, in tat protein of HIV, which is essential for replication in the virus, lysines and histidines that are essential to maintaining the Replikin definition within a key Replikin peptide in the protein are observed to be 100% conserved, while substitutions in amino acid residues that are not essential to maintaining the Replikin definition are commonly substituted. The conservation of the key amino acids for maintaining the Replikin definition is understood to provide a specific survival function for HIV. The same phenomenon is seen in influenza. See U.S. Pat. No. 7,442,761, column 62, lines 42-45.
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 50 amino acid residues comprising (1) a first lysine residue six to ten residues from a second lysine residue, (2) at least one histidine residue, (3) and 6% or more lysine residues where the Replikin sequence is the shortest sequence comprising the first and second lysine residues of element (1) and the at least one histidine residue of element (2). A Replikin sequence may comprise more than two lysine residues and more than one histidine residue so long as there is at least one lysine residue six to ten residues from a second lysine residue and at least one histidine residue. A Replikin sequence for the purpose of determining Replikin concentration as described in this paragraph may also be a nucleic acid that encodes a Replikin peptide sequence defined according to this paragraph.
Quantitative Real-Time Replikins Analysis of the virus genome of lethal Middle Eastern Respiratory Syndrome Coronavirus (MERS CoV) demonstrates in
The Figure summarizes all of the data listed in at the NCBI PubMed database. No data were available for large intervals of up to four months. Further, many sequences were published only up to 4 months after specimen collection.
The rapid quantitative decline in MERS CoV gene Replikin sequences reflected in
Because genomic Replikin changes can occur rapidly, and these correlate very closely with clinical outcomes, it is important that the genomic sequences be published as soon as possible after the specimens are collected. Ideal surveillance, attainable now, would have sequences published within a few days of specimen collection and Replikin analysis performed within an additional 24 hours. Testing completely synthetic Replikins vaccines based on this technology can be available quickly, in days to weeks.
One non-limiting aspect of the present invention provides a method of predicting expansion or retraction of a population of MERS CoV comprising, respectively, identifying an increase in the percentage of isolates of MERS CoV having a Replikin concentration (number of Replikin sequences per 100 amino acid residues) greater than 4.0 between two time periods or identifying a decrease in the percentage of isolates of MERS CoV having a Replikin concentration (number of Replikin sequences per 100 amino acid residues) greater than 4.0 between two time periods. In a non-limiting embodiment, the more than two time periods are compared and the percentage of isolates in the more than two time periods shows a pattern of increase or decrease.
In a further non-limiting embodiment, a plurality of isolates in a given time period may be analyzed for Replikin concentration. The percentage of isolates having a Replikin concentration of greater than 4.0 Replikin sequences per 100 amino acid residues in a first time period may be compared to the percentage of isolates having a Replikin concentration of greater than 4.0 Replikin sequences per 100 amino acid residue in a second time period. If the later timer period has a higher percentage of isolates having a Replikin concentration of greater than 4.0 then it is predicted that the MERS population will expand and an outbreak is more likely. If the later time period has a lower percentage of isolates having a Replikin concentration of greater than 4.0 then it is predicted that the MERS populations will retract and an outbreak is expected to decrease in severity.
For example,
One non-limiting aspect of the present invention provides a method of determining an increased probability of an outbreak of MERS CoV within about six months to about three years following an increase in Replikin concentration in an isolate of MERS CoV comprising identifying an increase in the concentration of Replikin sequences in at least one first isolate of MERS CoV as compared to at least one other isolate of MERS CoV wherein said at least one first isolate is isolated later than said at least one other isolate is isolated, and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of MERS CoV within about six months to about three years following the increase in the concentration of Replikin sequences. In a non-limiting embodiment, the first isolate of MERS CoV is isolated at least about six months later than the at least one other isolate.
In a non-limiting embodiment, a method of prediction comprises: (1) obtaining a plurality of isolates of MERS CoV, wherein at least one of said isolates is isolated later (less than six months later or about six months to about 3 years later) than at least one other of said isolates; (2) analyzing the amino acid sequence of at least one protein or protein fragment in each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (3) comparing the concentrations of Replikin sequences in the at least one protein or protein fragment in each isolate of the plurality of isolates one to another; (4) identifying an increase in the concentration of Replikin sequences in said plurality of isolates over at least one time period (said period may be one day, one week, one month, or six months or greater); and (5) predicting an outbreak of MERS CoV following said identified increase in the concentration of Replikin sequences. In one embodiment of the invention, the outbreak of MERS CoV is predicted within about six months to about five years. In a further embodiment of the invention, the outbreak of MERS CoV is predicted within about one year to about three years.
In a further non-limiting embodiment of the aspect of the invention, the method of prediction further comprises comparison of the standard deviation of the mean of Replikin concentrations of isolates of MERS CoV from a given time period, such as a given month, a given year, or any other given time period. In a further non-limiting embodiment the Replikin concentration is a mean Replikin concentration of a plurality of isolates with standard deviation from the mean and the standard deviation from the mean is greater than the standard deviation from the mean Replikin concentration of a plurality of other isolates.
A prediction of expansion or retraction of a MERS CoV population may be performed by a processor. A prediction may be output to a user or display. Likewise, a particular Replikin peptide or Replikin Peak Gene within an isolate or population of isolates of MERS CoV predicted to be expanding or retracting in replication or lethality may be output to a user or display. A machine-readable storage medium may contain executable instructions that, when executed by a processor, cause the processor to provide sufficient data to a user, a printout, or a display such that the user or a user of the printout or display may predict expansion or retraction of population of MERS CoV. A process for prediction may comprise: comparing a Replikin Count of at least one first isolate of MERS CoV with a Replikin Count of at least one second isolate of MERS CoV; and predicting the population of the first isolate to be expanding if the Replikin Count of the first isolate is greater than the Replikin Count of the second isolate. In another embodiment, predicting the population of the first isolate to be expanding if the Replikin Count of the first isolate is greater than four Replikin sequences per 100 amino acid residues.
A computer system may include a processor coupled to a network, and a memory coupled to a processor, wherein the memory contains a plurality of instruction to perform the methods of prediction discussed herein.
A user of outputted data from a processor, storage medium, machine-readable medium, signal, or computer system may include any person or any machine that records or analyzes the outputted data. A display or printout may include any mechanism by which data is outputted so that any person or any machine may record or analyze the outputted data, including a printed document, a visual impulse, an aural impulse, a signal, or any other perceivable impulse, a computer monitor, a set of numbers, or any other display or printout of data including a digital recording medium.
Concerning a non-limiting computer system that may implement methods disclosed in the application,
Once a string of amino acids is found to match the Replikin pattern, the string may be identified or marked (520) accordingly.
A given sequence of amino acids may contain many subsequences or strings that match the Replikin pattern. Additionally, Replikin patterns may overlap each other. Thus, to locate and identify all possible Replikin patterns in a sequence of amino acids, method 500 may be invoked iteratively for each subsequence of amino acids contained within the original sequence of amino acids.
When method 500 is invoked iteratively to identify and locate all possible Replikin patterns in an amino acid sequence, the number of resulting Replikin patterns may be counted. A Replikin count may be reported as an absolute number. Additionally, a ratio of the number of Replikins per N amino acids in the sequence may be calculated. For example, it may be determined that a given protein contains a ratio of 6 Replikin sequences for every 100 amino acid residues. Replikin ratios have been shown by laboratory experiment and by epidemiological evidence to correlate directly to the rate that a given virus replicates. Rapid replication in pathogens is an indication of disease. For example, the presence of relatively high ratios of Replikin patterns has been correlated to epidemics of influenza. Similarly, an increase in the count of Replikin patterns observed in a protein over time indicates a future disease caused by the organism from which the protein was obtained.
Once method 600 has identified two lysine residues that are close enough to each other (620), the method 600 may examine every histidine residue that resides within rmax positions of both the first and second lysine residues (630). When method 600 is employed to identify and locate typical Replikin patterns, rmax will be set to equal 50. For every histidine residue that resides within rmax positions of the two lysine residues identified in steps (610) and (620), method 600 will construct the shortest string of amino acid residues that includes the first lysine residue, the second lysine residue, and the identified histidine residue (640). Then, method 600 will determine whether the length of that shortest string is within the desired range—that is, whether it contains at least rmin amino acid residues and no more than rmax amino acid residues (650). Finally, if the identified string of amino acids also contains at least kpercent of lysine residues (660), the string will be identified as matching the desired Replikin pattern (670).
Still referring to
One non-limiting aspect of the present invention provides methods of predicting expansion or retraction of outbreaks of MERS CoV. Compounds for diagnostic, therapeutic, and/or preventive purposes in MERS CoV and therapies for the prevention and treatment of MERS CoV are provided based on the disclosed methods of prediction.
An aspect of the present invention provides compounds for diagnostic, therapeutic, and/or preventive purposes in MERS CoV, methods of differentiating relative lethality between one or more isolates of MERS COV, and methods of designing therapies against MERS CoV based on compounds of the invention and differentiation of lethality among isolates of MERS CoV.
Compounds of one aspect of the invention comprise Replikin peptides and homologues of Replikin peptides identified in and isolated from different strains of MERS CoV and include Replikin peptides conserved over time in the same and different strains of MERS CoV. These Replikin peptides are useful when comprised in immunogenic and blocking compounds to provide a protective effect against MERS CoV infection including antagonism of the lethality of strains of MERS CoV. Replikin peptides that are conserved within strains of MERS CoV over time or across different strains of MERS CoV at conserved positions in the different strains of MERS CoV provide the ordinary skilled artisan with an expectation that the functionality of these peptides share commonality among various strains of MERS CoV and among various isolates of the same strain of MERS CoV at different times.
Four peptides provided in an aspect of this invention were identified as conserved in isolates of MERS CoV. The four peptides are combined in a vaccine for administration against challenge by MERS CoV. The vaccine is designed to generate therapeutic blocking response and an immune response that antagonizes infectivity, replication, and transmission of MERS CoV. Any virus that is not blocked on entry will be blocked intracellularly.
The four peptides were surprisingly identified as conserved in MERS CoV across regions and time. The ORF4b gene area of MERS CoV has been shown to be a Replikin Peak Gene area of the virus and Replikin Peak Gene areas of other viruses and pathogens have been demonstrated to be associated with lethality. See, e.g., WO 2008/143717, FIGS. 10-13, 16, 17, and 19.
The peptides and their homologues described herein are, among other things, antigenic, common to various strains of MERS CoV in both position and function, and conserved in various strains of MERS CoV over time. One of ordinary skill in the art expects the Replikin peptides and their homologues described herein to be useful in immunogenic and blocking compounds for therapies against MERS CoV within strains, across strains, and across time.
Shared and Conserved Replikin Peptide Sequences and their Homologues
Replikin sequences and their homologues provided by an aspect of the invention may be identified in strains of MERS CoV including any strain of MERS CoV known now or identified or known hereafter. Compounds of the invention may be conserved within strains of MERS CoV, across types within strains of MERS CoV, and across strains of MERS CoV. Compounds of the invention may be conserved in humans and camels and may be preserved over one, two, or more years in these hosts. In a non-limiting embodiment, the Replikin sequences are shared among isolates isolated from camels and humans. In a non-limiting embodiment, the Replikin sequences are identical isolates isolated from camels and humans. In a non-limiting embodiment the Replikin sequences are identical and shared among camels and humans and are conserved among camels and humans. The compounds, because they are Replikin sequences, related to Replikin sequences, derived from Replikin sequences, identified as comprising Replikin sequences, or designed to comprise Replikin sequences, are related to rapid replication, virulence, and lethality in MERS CoV and comprise necessary structure for antigenicity. These characteristics of Replikin sequences have been previously established in other viruses and organisms (see, e.g., U.S. Pat. No. 7,894,999, U.S. Pat. No. 7,758,863 and WO 2008/143717) but have not previously been disclosed in MERS CoV; and the surprisingly effective utility of the Replikin sequence in predictions and therapies in MERS CoV is established herein. Compounds of the invention, including conserved Replikin peptides, are useful as immunogenic compounds to stimulate the immune system of a subject to produce an immune response, which may include production of antibodies or other binding molecules. Compounds of the invention are also useful in therapies such as vaccines. Compounds of the invention are likewise useful in producing antibodies, antibody fragments, or other binding or antagonizing agents, which may be used, among other things, for diagnostic and therapeutic purposes, including passive immunity. Conservation between camel reservoirs and human hosts allows for targeting of the Replikin structures in both hosts.
The immunogenic compounds, antibodies (and other binding or antagonizing agents) and vaccines of the invention are useful against any strain of MERS CoV and are likewise useful against other coronaviruses. The compounds of the invention are also useful for diagnostic purposes, including identifying rapidly replicating, virulent, or lethal strains of virus.
Information on the conservation of homologous sequences across various strains of MERS CoV and in different regions provides sequences that offer immunogenic compounds for antagonism of all strains comprising these homologues across all regions having strains comprising these homologues. As a result, a vaccine is provided herein that offers cross-strain protection for a variety of strains of MERS CoV.
Replikin peptides in general are seen to be conserved across strains of MERS CoV. The key amino acid residues that provide for the Replikin sequence structure are the lysine and histidine residues wherein a Replikin sequence has at least one lysine on one terminus and at least one lysine or one histidine on the other terminus, at least one lysine that is six to ten residues from at least one other lysine, at least one histidine, and at least six percent lysines in total between the terminal lysine and the terminal lysine or histidine.
As may be seen in FIG. 10 of WO 2005/104754, when conserved homologous Replikin sequences are aligned one on top of the other over time, it is most apparent that fixed and conserved portions of the structure of Replikin sequences align in a series of posts or girders that illustrate, like the structure of a building, how key conserved amino acids provide constancy for the survival of a virus such as MERS CoV over time as it mutates to avoid immune recognition in its prospective host but maintains key functional genetic structures that provide for continued replication of the virus. These key functional genetic structures provide targets antagonized by Replikin-based therapies.
One aspect of the present invention provides a protein, a protein fragment, a polypeptide, or a peptide that comprises at least one peptide A homologous with at least one Replikin peptide identified in an isolate of MERS CoV. The Replikin peptide may be any Replikin peptide identified in an isolate of MERS COV. The Replikin peptide may further be a Replikin peptide identified as conserved across strains or across regions in isolates of MERS COV or any homologue of a Replikin peptide identified as conserved across strains or across regions in isolates of MERS COV. For example, the Replikin peptide may be any one of SEQ ID NO(s): 1-28 or any homologue of any one of SEQ ID NO(s): 1-28.
Peptide A of the protein, protein fragment, polypeptide, or peptide may be 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous or 100% homologous with a Replikin peptide, including any of the peptides of SEQ ID NO(s): 1-28. A protein fragment or peptide may likewise be a peptide that consists of a peptide A that is homologous with a Replikin peptide of MERS COV, including any of SEQ ID NO(s): 1-28. A peptide consisting essentially of or consisting of a Replikin peptide of MERS COV, including any one of SEQ ID NO(s): 1-28, is also provided.
The amino acid sequence of the provided isolated or synthesized protein, protein fragment, polypeptide, or peptide may partially match an amino acid sequence of an expressed whole protein. At least one, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred, five hundred and fifty or more amino acid residues of the amino acid sequence of the expressed whole protein may not be present in the protein, protein fragment, polypeptide, or peptide. The amino acid sequence of an isolated or synthesized protein fragment, polypeptide, or peptide may also partially match the amino acid sequence of an expressed whole protein where at least one, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of at least one terminus of the amino acid sequence of the expressed whole protein is (are) not present at at least one terminus of said protein fragment, polypeptide, or peptide. Any additional number of amino acids may be situated on one or the other terminus or on both termini of the protein fragment, polypeptide, or peptide.
Because a Replikin peptide, such as SEQ ID NO(s): 1-28, is associated with rapid replication, infectivity, and/or lethality, in functional proteins in MERS CoV and because a Replikin peptide such as any one of SEQ ID NO(s): 1-28 is antigenic, inclusion of any Replikin peptide in a protein, protein fragment, polypeptide, or peptide does not negate the functional nature of the Replikin peptide. As such, antagonism of at least one Replikin peptide, including at least one of SEQ ID NO(s): 1-28 or a homologue of SEQ ID NO(s): 1-28 (with homology of 30% or greater) within a protein, protein fragment, polypeptide, or peptide would be expected to antagonize the replication, infectivity, and/or lethality of the protein fragment, polypeptide, or peptide.
A provided peptide may further be a peptide B of 7 to about 50 amino acid residues where peptide B contains a peptide A that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous or 100% homologous with any Replikin peptide, including one of SEQ ID NO(s): 1-28. A non-limiting peptide may further be a peptide A that is a Replikin peptide.
An isolated or synthesized protein, protein fragment, polypeptide, or peptide may consist of a peptide that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one of SEQ ID NO(s): 1-28 where the length of the peptide is no more than one, five, ten, twenty, thirty, forty, or fifty amino acid residues longer than a Replikin peptide of MERS COV including the sequence of SEQ ID NO(s): 1-28 with which it is homologous. An isolated or synthesized protein fragment, polypeptide, or peptide may likewise be no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues longer than the Replikin sequence with which it is homologous (including, for example, SEQ ID NO(s): 1-28).
Another non-limiting embodiment provides a biosynthetic composition of the invention. The biosynthetic composition may comprise the isolated or synthesized protein, protein fragment, polypeptide, or peptide of an aspect of the invention disclosed herein. The biosynthetic composition may further consist essentially of a Replikin peptide of a MERS CoV. In a further embodiment, the Replikin peptide of a MERS CoV is at least one peptide having a sequence of SEQ ID NO(s): 1-28. A further non-limiting embodiment provides a biosynthetic composition consisting of a Replikin peptide of MERS CoV. In a non-limiting embodiment, the isolated protein fragment, polypeptide, or peptide of an aspect of the invention is chemically synthesized by solid phase methods.
An isolated or synthesized polypeptide or peptide may comprise a peptide A that has about the same number of amino acid residues as a peptide B, where peptide B is one of the peptides of SEQ ID NO: 1-28 and where the lysine residues and histidine residues in peptide A are conserved as compared to the lysine residues and histidine residues in peptide B. An isolated or synthesized polypeptide or peptide comprising peptide A may have up to 100 additional amino acid residues as compared to peptide B. Some or all of the up to 100 additional amino acid residues may be positioned toward the amino-terminus and/or carboxy-terminus of the lysine or histidine termini of peptide A. Some of the additional amino acid residues may be positioned within the lysine or histidine termini of peptide A so long as a level of homology is maintained between peptide A and peptide B that retains at least some of the functionality of the Replikin peptide of peptide B. Functionality may include, but is not limited to, antigenicity, rate of replication, antagonizability of a protein containing said peptide A or said peptide B, binding capacity of binding agents to peptides A or B, etc.
An isolated or synthesized polypeptide or peptide may also comprise up to about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, about 4, about 3, about 2, or about 1 additional amino acid residues. The residues may be entirely outside of the Replikin structure or entirely within the Replikin structure or partially within and partially outside the Replikin structure. A level of homology should be maintained between peptides B and A when additional residues are present or are added. Residues outside of the Replikin structure are those residues on the amino-terminus or carboxy-terminus of the polypeptide or peptide as compared to the lysine or histidine termini of peptide A. Residues within the Replikin structure are those residues that are between the lysine or histidine termini of peptide A. An isolated or synthesized polypeptide or peptide may also consist of peptide A and peptide A may consist of peptide B.
The at least one isolated or synthesized protein, protein fragment, or peptide may also comprise at least one peptide A and at least one peptide C where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one Replikin peptide of MERS CoV, which may include a peptide of SEQ ID NO(s): 1-28, and where peptide C is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one other Replikin peptide of MERS CoV, which also may include a peptide of SEQ ID NO(s): 1-28. Peptide C may be homologous with a different Replikin peptide than the peptide that peptide A is homologous with. The at least one isolated or synthesized protein, protein fragment, or peptide may comprise three or more peptides homologous with at least three different Replikin peptides.
All of the above-discussed proteins, protein fragments, polypeptides, and peptides comprise the functional unit of a homologue of a Replikin peptide present in or isolated from an MERS CoV. The Replikin peptide may be any one of SEQ ID NO(s): 1-28 or any one of SEQ ID NO(s): 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. Antagonism of any of the homologues of a Replikin peptide will antagonize replication in MERS CoV. As a result, the proteins, protein fragments, polypeptides, and peptides are useful as immunogenic compounds, therapeutic compounds, vaccines, and for other therapies directed to antagonizing the replication and/or lethality of a strain of MERS CoV. When comprised in a vaccine, disclosed proteins, protein fragments, polypeptides, and peptides are expected to be capable of limiting the excretion or shedding of MERS CoV such that the virus is limited in its spread from host to host or from host to reservoir to host, etc. As such, disclosed compounds are effective at limiting sources of MERS CoV infection. Likewise, any binding agent that binds one of the proteins, protein fragments, polypeptides, and peptides discussed above will antagonize the replication and/or lethality of a strain of MERS CoV and limit sources of MERS COV infection such as transmission from host to host or from host to reservoir to host.
A protein, protein fragment, or peptide comprising a Replikin peptide present or identified in an isolate of MERS CoV may be comprised in an immunogenic compound. The proteins, protein fragment, polypeptides, and peptides provided by an aspect of the invention comprise at least a portion that is homologous with a Replikin peptide or homologous with one of the MERS CoV Replikin peptides of SEQ ID NO(s): 1-28. These homologues are expected by one of ordinary skill in the art to stimulate the immune system of a subject upon sufficient exposure to produce antibodies against at least the homologous portion of the protein, protein fragment, polypeptide, or peptide and/or to produce a protective effect against MERS CoV. One of ordinary skill in the art would expect that antibodies or other binding agents arrayed against a protein or protein fragment comprising one of the antigenic homologues disclosed herein would be antagonistic to the protein or protein fragment.
One of ordinary skill would also expect an antagonist of one of these homologues to antagonize any MERS CoV that comprises a homologue of a Replikin peptide. Because homologues of SEQ ID NO(s): 1-28 have been shown to be conserved across strains and time, one of ordinary skill would expect antagonism of such homologues to result in antagonism of MERS CoV replication. One of ordinary skill would further expect particular antagonism of the lethality mechanisms of MERS CoV when an immune system is stimulated against a homologue of any one or more of SEQ ID NO(s): 1-28.
As a result, one aspect of the present invention is a method of stimulating the immune system of a subject with at least one compound comprising at least one Replikin sequence identified in MERS CoV or at least one isolated or synthesized homologue or functional fragment of at least one Replikin sequence identified in MERS CoV. The at least one Replikin sequence of the compound reflects an immunogenic target against which the immune system of the subject responds. Because at least a functional portion of the immunogenic structure of the target is maintained in a functional fragment of the at least one Replikin sequence, a functional fragment of the Replikin sequence is likewise a target against which the immune system of the subject responds. The compound may comprise a protein comprising the at least one Replikin sequence or functional fragment thereof, a protein fragment, a polypeptide, or a peptide comprising the at least one Replikin sequence or functional fragment thereof. The compound may comprise more than one protein, protein fragment, polypeptide or peptide. The compound may further be a composition of a plurality of synthesized or isolated Replikin sequences.
An immunogenic compound provided as an aspect of the invention may be used as a component of a non-limiting vaccine against any strain of MERS CoV. A vaccine comprising one or more homologues of a Replikin peptide of MERS CoV may be used against MERS CoV. The vaccine may comprise one or more homologues of SEQ ID NO(s): 1-28 or 30-172, 174-190, 192-209, 211-222, 224-235, and 237-239. Likewise, a vaccine comprising one or more homologues of a Replikin peptide may be used against MERS CoV and may antagonize the replication and/or lethality of an MERS CoV infection. Further, mixtures of homologues of SEQ ID NO(s): 1-28 are provided as vaccines to antagonize the replication and/or lethality of an MERS CoV infection. Such vaccines are useful for antagonizing replication, lethality, and excretion or spread of MERS CoV.
One vaccine may comprise at least one protein, protein fragment, polypeptide, or peptide of any one or more of the proteins, protein fragments, polypeptides, or peptides of an aspect of the invention. The vaccine may further comprise at least one Replikin peptide of MERS CoV. One such Replikin peptide may be any one of SEQ ID NO(s): 1-28. A vaccine may comprise at least one peptide consisting essentially of any one of SEQ ID NO(s): 1-28 or at least one peptide consisting of any one of SEQ ID NO(s): 1-28. A vaccine may comprise a mixture of Replikin peptides of MERS CoV. The vaccine may comprise a plurality of peptides such as two, three, or four peptides of SEQ ID NO(s): 1-28. The peptide may consist essentially of any one or more SEQ ID NO(s): 1-28 or may consist of any one or more of SEQ ID NO(s): 1-28.
In another non-limiting embodiment, the vaccine comprises a mixture of peptides, wherein the mixture comprises isolated or synthesized peptides of SEQ ID NO(s): 1-9, SEQ ID NO(s):1-28, SEQ ID NO(s): 5, 18, 22, and 23, or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a non-limiting embodiment, the vaccine comprises an approximately equal molar mixture of the isolated or synthesized peptides of SEQ ID NO(s): 1-9, SEQ ID NO(s):1-28, SEQ ID NO(s): 5, 18, 22, and 23, or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. In a further non-limiting embodiment, the vaccine comprises approximately equal weight of the isolated or synthesized peptides of SEQ ID NO(s): 1-9, SEQ ID NO(s):1-28, SEQ ID NO(s): 5, 18, 22, and 23, or SEQ ID NO(s): 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28.
A vaccine may comprise a plurality of the shortest Replikin peptides from any region of the genome of MERS CoV including the ORF 4b gene area. A vaccine may comprise the shortest Replikin peptides from a gene area identified in a MERS CoV isolate or a plurality of MERS CoV isolates predicted to have a greater lethality than at least one other isolate of MERS CoV.
A vaccine may further comprise a plurality of the longest Replikin peptides from any gene area of the virus including the ORF4b gene area identified in an MERS CoV isolate or a plurality of MERS CoV isolates. A vaccine may also comprise a mixture of the shortest and longest Replikin peptides identified in the ORF4b gene area or any other gene area.
A vaccine may be formulated with a pharmaceutically acceptable excipient, carrier, or adjuvant. One pharmaceutically acceptable carrier or excipient is sterile water. A vaccine may comprise freeze-dried Replikin peptides in sterile water. Excipients, carriers, or adjuvants may include, but are not limited to, excipients, carriers and adjuvants known to those of skill in the art now or hereafter.
A non-limiting acceptable carrier or adjuvant may include sterile water, oil and water emulsion, or keyhole limpet hemocyanin, A non-limiting carrier and/or adjuvant may include a sterile diluent such as water (for dermal, nasal, or ocular application, spraying, or injection), saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for use typically include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In general, a relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof
In a further non-limiting embodiment, one adjuvant is a UTOPE. A UTOPE may be covalently attached to an isolated or synthesized peptide at the C-terminus, the N-terminus, or both termini. A UTOPE is a peptide sequence of 6 to 10 residues comprising one histidine residue with all other residues being lysine residues. A UTOPE may be included in a vaccine for the treatment or prevention of MERS CoV infection.
Peptides may be synthesized using L- or D-amino acids and may be synthesized with chemical moieties to discourage breaking of peptide bonds in biological systems.
A composition of the invention may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal or any other routes. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
Administration of the vaccine via any method may produce an immune response in an animal or human, it may further produce an antibody response in the animal or human. In a further non-limiting embodiment, the vaccine may produce a protective effect in the animal or human.
Generally, the dosage of peptides is in the range of from about 0.01 μg to about 500 mg, from about 0.05 μg to about 200 mg, about 0.075 μg to about 30 mg, about 0.09 μg to about 20 mg, about 0.1 μg to about 10 mg, about 10 μg to about 1 mg, and about 50 μg to about 500 μg. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response and/or blocking effect.
A non-limiting composition is provided comprising one or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-28. A composition is provided comprising one or more isolated or synthesized peptides consisting essentially of or consisting of at least one peptide of SEQ ID NO(s): 1-28. A composition is further provided comprising two, three, four five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or up to twenty-eight isolated or synthesized peptides of SEQ ID NO(s): 1-28.
A composition comprising a mixture of peptides is provided wherein the mixture comprises at least each of the isolated or synthesized peptides of SEQ ID NO(s): 1-9; 1-28; 5, 18, 22, and 23; or 6, 11, 12, 13, 14, 18, 19, 20, 26, 27, and 28. A mixture is provided that is equimolar. A mixture is also provided that is equal by weight.
A composition of an aspect of the invention is a therapeutic composition. The therapeutic composition may provide, upon administration to a subject, a therapeutic effect against MERS COV. The therapeutic composition may be administered to any animal susceptible to infection from MERS CoV. The therapeutic composition may be administered to any animal susceptible to MERS CoV, including a human or a camel.
Identification of conserved Replikin peptides across strains of MERS CoV in different countries has provided for the development of vaccines that may be directed across strains of MERS CoV in different countries. Identification of conserved Replikin peptides in isolates of MERS CoV of any strain may be accomplished in any way known to one of skill in the art now or hereafter. One method is by review of in silico sequences provided at the PubMed website of the National Center for Biotechnology Information. Peptides that share exact identity or 100% homology with earlier identified Replikin peptides may be tracked using computer-searching methods. Peptides that share 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homology with an earlier identified Replikin peptide may also be tracked by computer methods.
For example, vaccines have now been developed for prevention and treatment of infection from MERS CoV. See, e.g., Examples 1-3 below. The sequences that are used in the vaccine in Example 1 are identically shared among human and camel hosts and conserved from 2012 to 2014. The vaccine in Example 2 likewise comprises sequences shared and conserved. The sequences that are used in the vaccine in Example 3 have now been identified as conserved across countries and strains of MERS CoV. All of these sequences, homologues of these sequences, and proteins, protein fragments, polypeptides, and peptides comprising, consisting essentially of, or consisting of these sequences or their homologues or functional fragments are useful in identifying lethal strains of MERS COV, treating infections from MERS CoV, and developing prophylactic therapies, such as vaccines, against infection from MERS CoV.
These proteins, fragment, polypeptides and peptides including any one of SEQ ID NO(s): 1-28 and/or homologues of any one of SEQ ID NO(s): 1-28 and/or functional fragments of SEQ ID NO(s): 1-28 are expected by one of ordinary skill in the art to provide antigenicity, antagonism of replication, and blocking that is comparable to any one of SEQ ID NO(s): 1-28.
Homology that is sufficient to produce a useful target for antagonism includes peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100% homologous with any of SEQ ID NO(s): 1-28. Homology may be determined with peptides wherein gaps exists in the sequence that is being compared to any one of SEQ ID NO(s): 1-28 between amino acids that are identical to those of the peptide chosen from SEQ ID NO(s): 1-28.
Sequences that are conserved across strains of MERS CoV are excellent targets for controlling infectivity and lethality.
On aspect of the invention also provides methods of designing and making vaccines. For example, the invention provides a method of making a vaccine comprising selecting at least one or more isolated or synthesized Replikin peptides present or identified in an isolate of MERS CoV. Such peptides may include any one or more of SEQ ID NO(s): 1-28 as a component of a vaccine and making said vaccine. The method may comprise selecting from 1 to up to 4 or more isolated or synthesized MERS CoV Replikin peptides as a component of a vaccine. The peptides may be identified in the ORF4b gene area of the virus or may be identified in any area of the genome of MERS CoV. The method may comprise identifying one or more Replikin peptides in an emerging strain of MERS CoV up to about three years before the vaccine is administered, up to about one year before the vaccine is administered, up to about six months before the vaccine is administered, or up to about seven days before the vaccine is made.
An emerging strain may be any strain of MERS CoV identified by one of skill in the art as predicted to expand in a population in hosts, or predicted to increase in virulence, morbidity, and/or mortality (lethality) in its hosts. An emerging strain may likewise be a strain of MERS CoV wherein Replikin concentration is observed to be increasing over time. An emerging strain may likewise be a strain of MERS CoV identified within a rising portion of Replikin cycle, following a peak in a Replikin cycle, following a step-wise rise in a Replikin cycle, or identified by a Replikin Count Virus Expansion Index as an emerging strain of virus. See WO 2009/132209, the contents of which are incorporated herein by reference.
A method of making a vaccine is also provided comprising: selecting at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising a homologue of a Replikin peptide (including, for example, SEQ ID NO(s): 1-28) as a component of a vaccine; and making said vaccine. An isolated or synthesized protein, protein fragment, polypeptide, or peptide may comprise a peptide that is 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one Replikin peptide. At least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more homologues of Replikin peptides may be selected. Also, at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more Replikin peptides may be selected. The isolated or synthesized protein, protein fragment, polypeptide, or peptide may have the same amino acid sequence as at least one protein, protein fragment, polypeptide or peptide identified in an emerging strain of MERS COV up to one, two, or three or more years prior to making said vaccine. The at least one protein, protein fragment, polypeptide or peptide may be identified in an emerging strain of MERS COV one week, one month, two months, three months, four months, five months, or six months prior to making said vaccine.
The invention also provides a kit for making a vaccine where the kit includes at least one isolated or synthesized Replikin peptide of MERS CoV (including, for example, at least one peptide of SEQ ID NO(s): 1-28 or homologue of SEQ ID NO(s): 1-28). The kit may also include two, three, four, and up to twenty-eight or more peptides of SEQ ID NO(s): 1-28 or homologues of SEQ ID NO(s): 1-28.
In another aspect of the invention, isolated Replikin peptides may be used to generate antibodies, antibody fragments, or to generate or identify other binding agents, which may be used, for example, for diagnostic purposes or to provide passive immunity in an individual. See, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 and U.S. application Ser. No. 12/010,027, filed Jan. 18, 2008 (each incorporated herein by reference in their entirety).
Various procedures known in the art may be used for the production of antibodies to Replikin sequences or to proteins, protein fragments, polypeptides, or peptides comprising Replikin sequences. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that are linked to a cytotoxic agent or signaling moiety may also be generated. Antibodies may also be administered in combination with an antiviral agent. Furthermore, combinations of antibodies to different Replikins may be administered as an antibody cocktail.
For the production of antibodies, various host animals may be immunized by injection with a Replikin peptide or a combination of Replikin peptides, including, but not limited to rabbits, mice, rats, and larger mammals. Monoclonal antibodies to Replikins may be prepared using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) or other techniques may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Replikin-specific single chain antibodies. Antibody fragments that contain binding sites for a Replikin may be generated by known techniques. For example, such fragments include but are not limited to F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecules and or fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments. Alternatively, Fab expression libraries can be generated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
Binding agents are provided including an antibody, antibody fragment, or binding agent that binds to at least a portion of an amino acid sequence of at least one protein, protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one Replikin peptide of MERS CoV, which may include, for example, at least one Replikin peptide of SEQ ID NO(s): 1-28. The amino acid sequence of a protein fragment, polypeptide, or peptide may partially match the amino acid sequence of an expressed whole protein where at least one, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred or more amino acid residues of the amino acid sequence of the expressed whole protein are not present in the protein fragment, polypeptide, or peptide. The amino acid sequence of the protein fragment, polypeptide, or peptide may also partially match the amino acid sequence of an expressed whole protein where at least one, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of the amino acid sequence of at least one terminus of the expressed whole protein are not present at least one terminus of said protein fragment, polypeptide, or peptide.
Binding agents are also provided including an antibody, antibody fragment, or binding agent that binds to at least a portion of an amino acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one Replikin peptide of MERS CoV. In a non-limiting embodiment, the length of peptide A may be no more than one, five, ten, twenty, thirty, forty, or fifty amino acid residues longer than the identified Replikin sequence with which it is homologous. Binding agents are also provided that bind to at least a portion of an amino acid sequence of at least one of SEQ ID NO(s): 1-28.
Binding agents may specifically or preferentially bind to the target protein, protein fragment, polypeptide, or peptide. Binding agents may specifically or preferentially bind to a homologue of at least one of SEQ ID NO(s): 1-28. Binding agents may likewise specifically or preferentially bind to a peptide consisting of any one of SEQ ID NO(s): 1-28. Binding agents may also specifically or preferentially bind to a portion of a peptide consisting of any one of SEQ ID NO(s): 1-28 including a single amino acid within a homologue of SEQ ID NO(s): 1-28, two amino acids, three amino acids, four amino acids, five amino acids, or any number of amino acids spread within or outside a homologue.
An isolated or synthesized nucleic acid sequence is also provided that encodes a protein, protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one Replikin peptide of MERS CoV. The at least one Replikin peptide may be any peptide of SEQ ID NO(s): 1-28. A nucleic acid sequence may also encode a protein, a protein fragment, a polypeptide, or a peptide where the amino acid sequence of the protein, protein fragment, polypeptide, or peptide partially matches the amino acid sequence of an expressed whole protein and at least one, two, three, four, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred or more amino acid residues of the amino acid sequence of the expressed whole protein are not present in the protein fragment, polypeptide, or peptide. Further, the amino acid sequence of the protein, protein fragment, polypeptide, or peptide may partially match the amino acid sequence of an expressed whole protein where at least one, two, three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of the amino acid sequence of at least one terminus of the expressed whole protein may not be present at least one terminus of the protein, protein fragment, polypeptide, or peptide
An isolated or synthesized nucleic acid sequence may also encode a peptide consisting of 7 to about 50 amino acid residues comprising at least one Replikin peptide, which may be one of the peptide sequences of SEQ ID NO(s): 1-28. It may also encode a peptide that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptide sequences of SEQ ID NO(s): 1-28. It may also encode a peptide consisting essentially of or consisting of at least one of the peptide sequences of SEQ ID NO(s): 1-28.
One aspect of the invention further provides an immunogenic composition that comprises an isolated or synthesized nucleic acid provided above. One aspect of the invention further provides a vaccine against MERS CoV comprising an isolated or synthesized nucleic acid provided above.
Anti-Sense Nucleic Acids and siRNA
One aspect of the invention further provides a nucleic acid sequence that is antisense to a nucleic acid that encodes for any Replikin peptide present in or identified in an MERS CoV isolate. This may include one of SEQ ID NO(s): 1-28 or a small interfering nucleic acid sequence that interferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes any Replikin peptide of MERS CoV including, for example, any one of SEQ ID NO(s): 1-28 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-28.
The nucleotide sequence of the invention may be used in hybridization assays of biopsied tissue or blood, e.g., Southern or Northern analysis, including in situ hybridization assays, to diagnose the presence of a particular MERS CoV strain in a tissue sample or an environmental sample, for example. The present invention also provides kits containing antibodies specific for particular Replikins that are present in a particular isolate of MERS CoV, or containing nucleic acid molecules (sense or antisense) that hybridize specifically to a particular Replikin sequence, and optionally, various buffers and/or reagents needed for diagnosis.
Also within the scope of an aspect of the invention are oligoribonucleotide sequences that include antisense RNA and DNA molecules and ribozymes that function to inhibit the translation of Replikin-containing mRNA. Both antisense RNA and DNA molecules and ribozymes may be prepared by any method known in the art. The antisense molecules can be incorporated into a wide variety of vectors for delivery to a subject. The skilled practitioner can readily determine the best route of delivery, although generally intravenous or intramuscular delivery is routine. The dosage amount is also readily ascertainable.
An aspect of the invention further provides antisense nucleic acid molecules that are complementary to a nucleic acid of the invention, wherein the antisense nucleic acid molecule is complementary to a nucleotide sequence encoding a peptide of the invention. In particular the nucleic acid sequence may be anti-sense to a nucleic acid sequence that has been demonstrated to be conserved over a period of six months to one or more years and/or which are present in a strain of MERS CoV shown to have an increase in concentration of Replikin sequences relative to Replikin concentration in other MERS CoV strains.
An aspect of the invention also provides compositions comprising RNAi-inducing entities used to inhibit or reduce MERS CoV infection or replication including small interfering RNA, which is a class of about 10 to about 50 and often about 20 to about 25 nucleotide-long double-stranded RNA molecules. siRNA is involved in the RNA interference pathway, where it interferes with the expression of a specific gene such as the ORF4b gene area of MERS CoV. siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism.
An effective amount of an RNAi-inducing entity is delivered to a cell or organism prior to, simultaneously with, or after exposure to MERS CoV. A dosage may be sufficient to reduce or delay one or more symptoms of MERS CoV infection. Compositions of the invention may comprise a single siRNA species targeted to a target transcript or may comprise a plurality of different siRNA species targeting one or more target transcripts.
One aspect of the invention provides a small interfering nucleic acid sequence that is about 10 to about 50 nucleic acids in length and is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes for any portion of an MERS CoV Replikin peptide including, for example, any portion of SEQ ID NO(s): 1-28 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any portion of a Replikin peptide, including, for example, a portion of one of SEQ ID NO(s): 1-28. In a further non-limiting embodiment, the nucleic acid sequence is about 15 to about 30 nucleic acids. In a further non-limiting embodiment, the nucleic acid sequence is about 20 to about 25 nucleic acids. In a further non-limiting embodiment, the nucleic acid sequence is about 21 nucleic acids.
Advance information concerning Replikin peptides in expanding strains of pathogen allows for the rapid production of specific effective synthetic vaccines using one, or a combination, of Replikin peptides. Such synthetic vaccines have been demonstrated in rabbits, chickens, and shrimp. See, e.g., Examples 1-3 herein, Examples 6 and 7 of U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006, and Example 2 of U.S. application Ser. No. 12/108,458, filed Apr. 23, 2008. For example, a mixture of Replikin peptides administered orally to shrimp provided up to a 91% protective effect for shrimp challenged with taura syndrome virus. Taura syndrome virus is an often-lethal rapidly replicating pathogen that has a significant negative impact on the shrimp industry.
Synthetic Replikin vaccines have also been demonstrated in the H5N1 strain of influenza virus in chickens. For example, in a test of chickens administered a mixture of twelve H5N1 Replikin peptides from the hemagglutinin and pB1 gene areas intranasally, intraocularly, and by spray inhalation and challenged with low pathogenic H5N1 influenza isolated from a black duck in the state of North Carolina in the United States, a protective effect was observed at both the entry site of influenza (diminished antibody production in the serum 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 1 of U.S. application Ser. No. 12/581,112, filed Oct. 16, 2009 (incorporated herein by reference).
Administration of Replikin peptides in both shrimp and chickens appears to have provided a notable measure of mucosal immunity. For example, in Example 2 of U.S. application Ser. No. 12/108,458, a mixture of Replikin peptides was administered by mouth to shrimp later challenged with taura syndrome virus. The 91% protective effect of the vaccine is expected to have been a result, at least in part, of a mucosal immune-like response in the gut of the shrimp.
Likewise, in chickens, the administration of a mixture of Replikin peptides provided a protective effect against entry of the H5N1 virus. For example, as may be seen in Example 1 of U.S. application Ser. No. 12/581,112, filed Oct. 16, 2009 (incorporated herein by reference), 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' system. Some virus apparently entered the system of the chickens but was then blocked intracellularly. While the applicants do not wish to be bound by theory, the virus may have been blocked in its intracellular transport to the RNA or in synthesis of virus on RNA or in transport from the RNA to excretion. Wherever the block occurs, the fact is that the examination of the excreta of the chicken showed complete absence of virus. For those three chickens in which a serum immune response was measured (that is, virus did enter their system), 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.
A synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of nine MERS CoV Replikin peptides was designed for use against relatively-lethal isolates of MERS CoV. The sequences were found to be identical, shared Replikin structures in MERS CoV in dromedary camels (Camelus dromedaries) and humans. The vaccine was designed as a best-fit synthetic vaccine against MERS CoV for identified isolates from 2012 to 2014 shared among these two host species. The vaccine makes possible immediate MERS-CoV Replikins Synthetic Vaccine™ tests in camels and rapid interruption of the spread of the MERS-CoV coronavirus, more lethal to humans than SARS 2003.
The vaccine was engineered to inhibit the lethality of relatively-lethal strains of MERS CoV and to attack the camel reservoir of the virus as well as protect humans.
The vaccine comprises a mixture of the following nine Replikin peptides in sterile water:
The vaccine is administered to a camel and an immune response is detected. In camels where some antibody is detected prior to administration of the vaccine, administration of the vaccine produces an increase in antibody response and an increase in immune protection. Challenge of the camel with MERS-CoV isolates results in a protective effect against the challenge including reduction in entrance of virus into camel at inoculation, reduction in replication of virus in camel system, and blocking of excretion of virus from camel in body fluids. The camel is protected from infection. The vaccine is likewise tested in humans, providing an immune response, a blocking effect, and a protective effect.
The vaccine may be administered to an animal or human susceptible, exposed to, or suffering from infection of MERS CoV. The blocking mechanism of the vaccine provides a therapeutic and prophylactic effect. The immune response generated by the vaccine provides a prophylactic effect. Because each of the sequences comprised in the vaccine is related to rapid replication and lethality of the virus and provides an immune and a blocking response upon administration to a subject at sufficient volume and concentration, each sequence may be used as an individual active component in a vaccine against MERS-CoV; a mixture of peptides is not necessary to provide an effective vaccine.
A synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of twenty-eight MERS CoV Replikin peptides was designed for use against relatively lethal isolates of MERS CoV. The sequences were found to be shared Replikin structures in MERS CoV in dromedary camels (Camelus dromedaries) and humans. The vaccine was designed as a synthetic vaccine against MERS CoV for identified isolates from 2012 to 2014 shared among these two host species. The vaccine makes possible immediate MERS-CoV Replikins Synthetic Vaccine™ tests in camels and rapid interruption of the spread of the MERS-CoV coronavirus, more lethal to humans than SARS 2003.
The vaccine was engineered to inhibit the lethality of relatively-lethal strains of MERS CoV.
The vaccine comprises a mixture of the following twenty-eight Replikin peptides in sterile water:
The vaccine is administered to a camel and an immune response is detected. Challenge of the camel with MERS-CoV isolates results in a protective effect against the challenge including reduction in entrance of virus into camel at inoculation, reduction in replication of virus in camel system, and blocking of excretion of virus from camel in body fluids. The camel is protected from infection. The vaccine is likewise tested in humans, providing an immune response, a blocking effect, and a protective effect.
The vaccine may be administered to an animal or human susceptible, exposed to, or suffering from infection of MERS CoV. The blocking mechanism of the vaccine provides a therapeutic and prophylactic effect. The immune response generated by the vaccine provides a prophylactic effect. Because each of the sequences comprised in the vaccine is related to rapid replication and lethality of the virus and provides an immune and a blocking response upon administration to a subject at sufficient volume and concentration, each sequences may be used as an individual active component in a vaccine against MERS-CoV; a mixture of peptides is not necessary to provide an effective vaccine.
A synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of four MERS CoV Replikin peptides was designed for use against relatively lethal isolates of MERS CoV. The vaccine was engineered from sequences confirmed to be conserved across regions (countries) and across time. Conservation was particularly noted in the key amino acid residues of the Replikin sequence, namely, lysine and histidine amino acid residues. The vaccine was engineered to inhibit the lethality of relatively lethal strains of MERS CoV.
The vaccine comprises a mixture of the following four Replikin peptides in sterile water:
The vaccine may be administered to an animal or human susceptible, exposed to, or suffering from infection of MERS CoV. The blocking mechanism of the vaccine provides a therapeutic and prophylactic effect. The immune response generated by the vaccine provides a prophylactic effect.
106 a107 y108 s109 s110 s111 a112 n113 g114 s115 l116 v117 g118 t119 t120 l121 q122 g123 k124 p125 i126 g127
The following sequences are provided as examples of sequences conserved in isolates of MERS CoV across time and in various regions. Each sequence is provided below with a listing of the accession number and position in the publicly-disclosed sequence wherein the peptide sequence begins.
2012
AGV08591 position 247, AGV08415 position 247, AGV08386 position 247, YP_007188586 position 247, AFY13314 position 247, AGG22549 position 247, AGH58724 position 247, AFS88943 position 247.
2013
AGV08580 position 247, AGV08565 position 247, AGV08553 position 247, AGV08542 position 247, AGV08531 position 247, AGV08517 position 247, AGV08512 position 247, AGV08502 position 247, AGV08499 position 247, AGV08487 position 247, AGV08474 position 247, AGV08462 position 247, AGV08451 position 247, AGV08433 position 247, AGV08418 position 247, AGV08397 position 247, K9N4V7 position 247, AGN72646 position 247, AGN70980 position 247, AGN70970 position 247, AGN70958 position 247, AGN70936 position 247.
2012
AGV08583 position 1568, AGV08582 position 1568, AGV08407 position 1568, AGV08406 position 1568, AGV08378 position 1568, AGV08377 position 1568, YP 007188577 position 1568, YP 007188578 position 1568, AFY13306 position 1568, AFY13305 position 1568, AGG22541 position 1568, AGG22540 position 1568.
2013
K9N638 position 1568, K9N7C7 position 1568, AGV08596 position 1568, AGV08595 position 1568, AGV08572 position 1568, AGV08571 position 1568, AGV08557 position 1568, AGV08556 position 1568, AGV08555 position 155, AGV08545 position 1568, AGV08544 position 1568, AGV08534 position 1568, AGV08533 position 1568, AGV08522 position 241, AGV08491 position 1568, AGV08490 position 1568, AGV08479 position 1568, AGV08478 position 1568, AGV08466 position 1568, AGV08465 position 1568, AGV08454 position 1568, AGV08453 position 1568, AGV08443 position 1568, AGV08442 position 1568, AGV08436 position 525, AGV08402 position 156, AGV08389 position 1568, AGV08388 position 1568, AGN72639 position 1568, AGN72638 position 1568, AGN70972 position 1568, AGN70971 position 1568, AGN70961 position 1568, AGN70960 position 1568, AGN70950 position 1568, AGN70949 position 1568, AGN70928 position 1568, AGN70927 position 1568.
2012
AGV08587 position 23, AGV08411 position 23, AGV08382 position 23, YP 007188582 position 23, AFY13310 position 23, AGG22545 position 23, AFS88939 position 23.
2013
AGV08576 position 23, AGV08561 position 23, AGV08549 position 23, AGV08538 position 23, AGV08527 position 23, AGV08508 position 23, AGV08495 position 23, AGV08483 position 23, AGV08470 position 23, AGV08458 position 23, AGV08447 position 23, AGV08441 position 23, AGV08429 position 23, AGV08423 position 23, AGV08393 position 23, K9N643 position 23, AGN72642 position 23, AGN70976 position 23, AGN70965 position 23, AGN70954 position 23, AGN70932 position 23.
2012
AGV08587 position 20, AGV08411 position 20, AGV08382 position 20, YP 007188582 position 20, AFY13310 position 20, AGG22545 position 20, AFS88939 position 20.
2013 AGV08576 position 20, AGV08561 position 20, AGV08549 position 20, AGV08538 position 20, AGV08527 position 20, AGV08508 position 20, AGV08495 position 20, AGV08483 position 20, AGV08470 position 20, AGV08458 position 20, AGV08447 position 20, AGV08441 position 20, AGV08429 position 20, AGV08423 position 20, AGV08393 position 20, K9N643 position 20, AGN72642 position 20, AGN70976 position 20, AGN70965 position 20, AGN70954 position 20, AGN70932 position 20.
Conserved sequences are useful as targets for control of replication and lethality in vaccines. A vaccine combining conserved sequences or employing individual sequences or conserved sequences as vaccine targets may be used to prevent or control MERS CoV infection and outbreaks.
Gene and amino acid sequences publicly available for isolates of MERS CoV were queried at www.pubmed.com. Replikin concentrations were determined and numbers of isolates with Replikin concentrations greater than 4.0 were compared to numbers of isolates with Replikin concentrations 4.0 or less. A percentage was determined for each time period.
Table 1 provides the data from April 2012.
The number of isolates from April 2012 was 15. The number of isolates with a Replikin concentration of greater than 4.0 was 3. The percentage of isolates with a Replikin concentration of greater than 4.0 is 20%.
Table 2 provides the data from May through June 2012.
The number of isolates from May through June 2012 was 8. The number of isolates with a Replikin concentration of greater than 4.0 was 1. The percentage of isolates with a Replikin concentration of greater than 4.0 is 12.50%.
Table 3 provides the data from September 2012.
The number of isolates from September 2012 was 30. The number of isolates with a Replikin concentration of greater than 4.0 was 9. The percentage of isolates with a Replikin concentration of greater than 4.0 is 30%.
Table 4 provides the data from October 2012.
The number of isolates from October 2012 was 11. The number of isolates with a Replikin concentration of greater than 4.0 was 3. The percentage of isolates with a Replikin concentration of greater than 4.0 is 27%.
Table 5 provides the data from February 2013.
The number of isolates from February 2013 was 15. The number of isolates with a Replikin concentration of greater than 4.0 was 2. The percentage of isolates with a Replikin concentration of greater than 4.0 is 13.30%.
Table 6 provides the data from April 2013.
The number of isolates from April 2013 was 11. The number of isolates with a Replikin concentration of greater than 4.0 was 2. The percentage of isolates with a Replikin concentration of greater than 4.0 is 18.2%.
Table 7 provides the data from May 1, 2013.
The number of isolates from May 1, 2013 was 19. The number of isolates with a Replikin concentration of greater than 4.0 was 4. The percentage of isolates with a Replikin concentration of greater than 4.0 is 21.1%.
Table 8 provides the data from May 2-30, 2013.
The number of isolates from May 2-30, 2013 was 48. The number of isolates with a Replikin concentration of greater than 4.0 was 7. The percentage of isolates with a Replikin concentration of greater than 4.0 is 14.6%.
Table 9 provides the data from June 2013.
The number of isolates from June 2013 was 10. The number of isolates with a Replikin concentration of greater than 4.0 was 2. The percentage of isolates with a Replikin concentration of greater than 4.0 is 20%.
Accession number AHY22523 was analyzed. The following sequences were identified and with further analysis were determined to be conserved among camels and humans. AHY22523 is entitled “Middle East Respiratory Syndrome Coronavirus Quasispecies That Include Homologues of Human Isolates Revealed through Whole-Genome Analysis and Virus Cultured from Dromedary Camels in Saudi Arabia Direct Submission.”
A synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of the eleven MERS CoV Replikin peptides was designed for use against relatively lethal isolates of MERS CoV. The vaccine was engineered from sequences confirmed to be conserved among camels and humans across time. Conservation was particularly noted in the key amino acid residues of the Replikin sequence, namely, lysine and histidine amino acid residues. The vaccine was engineered to inhibit the lethality of relatively lethal strains of MERS CoV.
The vaccine may be administered to an animal or human susceptible, exposed to, or suffering from infection of MERS CoV. The blocking mechanism of the vaccine provides a therapeutic and prophylactic effect. The immune response generated by the vaccine provides a prophylactic effect. Infection and replication are blocked by targeting these sequences.
All isolates of the virus genes reported to date in at the NCBI PubMed database from both humans and camels was undertaken and revealed specific shared Replikin structures which were not just homologous but identical in the virus genes infecting both human and camel hosts, conserved since 2012 in cases from all countries affected. A Replikins Synthetic Vaccine based on these identical shared structures was produced. This vaccine is an inhibitor of the progression of MERS-CoV. The Anti-MERS-CoV vaccine was prepared for testing in humans and camels.
Camels are understood to be a reservoir and source of human MERS infection mostly because of the detection in camels of antibodies that react with MERS (1,2). The present quantitative gene specific structural findings support this understanding.
SARS, a coronavirus related to MERS, produced approximately 8000 human cases and 800 deaths in 2003. MERS has a higher mortality rate than SARS. MERS started in 2012 with an approximate mortality rate of 40%, and now, perhaps with better care, is still approximately 27%. The duration of the outbreak is already greater. This is in contrast to SARS, who's abrupt rise and rapid decline in gene Replikin concentration signaled in 2002 that the 2003 outbreak would occur but would be over soon (and it was within one year). The outbreak of MERS in 2012, the increased Replikin concentrations reported in October 2013, and the upsurge in MERS clinical outbreaks in 2014 now indicate a more prolonged course and a greater risk of pandemic. In agreement with these differences, the highest individual gene Replikin concentrations observed to date in MERS is double that in SARS.
The Replikins Synthetic Anti-MERS Vaccine is unique because:
Replikin sequences, therefore, provide a complete defense system, not previously available, beginning with prediction pre-outbreak, based on detection and specific identification of the quantitative gene changes associated with rapid replication. These gene changes can be addressed in advance of, or after, outbreaks occur, providing time to possibly prevent pandemics rather than just react to them.
While it would have been preferable to begin testing the Replikins vaccine when the upturn in gene Replikin Count was noted in October 2013, there still may be time to reduce or interrupt the present progression of MERS-CoV.
1. CIDRAP NEWS. Reports: Mers-CoV found in Saudi's patient's camel. Nov. 11, 2013.
2. Memish, Ziad. Promed Mail. Animal reservoirs, camels. http://www.isid.org Nov. 12, 2013.
3. Bogoch S and Bogoch ES. Nature Precedings doi: 10.1038/npre.2012.6952.1 Bogoch Replikins Pandemic Prevention: Increase of Strain-Specific Influenza Genomic Replikin Counts, Having Predicted Outbreaks and their Location Seven Times Consecutively, Up to Two Years in Advance, Provides Time for Prevention of Pandemics.
In October 2013, the applicants identified an increase in Replikin concentration in isolates of MERS-CoV.
The BBC reported the inventor's analysis that Middle East Respiratory Syndrome corona virus (MERS) has undergone several fluctuations in the virus' ability to replicate and could be rising again. The MERS Replikin concentration peaked in virulence and infectivity in September of 2012 with a count of 27.7% and dropped sharply to a low of 14.3% by February of 2013. The count began to rise again and as of June 2013 (the last month for which data were available at the time of analysis in October 2013) had reached 20%. “MERS Replikin Count Increases; Updated Data Needed,” BBC News Blog, Monday, Oct. 21, 2013, Michelle Blowers 3:56 p.m.
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| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/60906 | 10/16/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61891677 | Oct 2013 | US | |
| 61993332 | May 2014 | US |
